2,3-Dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercular agents: Design, synthesis, biological evaluation and SAR studies

Article abstract of DOI:10.1016/j.ejmech.2011.03.002

The alarming resurgence of tuberculosis (TB) underlines the urgent need for development of new and potent anti-TB drugs. Towards this goal we herein report the design and synthesis of 2,3-dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercula

Full text of DOI:10.1016/j.ejmech.2011.03.002

European Journal of Medicinal Chemistry 46 (2011) 2217e2223
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2,3-Dideoxy hex-2-enopyranosid-4-uloses as promising new anti-tubercular
agents: Design, synthesis, biological evaluation and SAR studies
Mohammad Saquib a, Irfan Husain a, Smriti Sharma a, Garima Yadav b, Vipul K. Singh b,
Sandeep K. Sharma b, Priyanka Shah c, Mohammad Imran Siddiqi c, Brijesh Kumar d, Jawahar Lal e,
**
,
a,
*
Girish K. Jain e, Brahm S. Srivastava b, Ranjana Srivastava b , Arun K. Shaw
a Medicinal and Process Chemistry Division, Central Drug Research Institute (CDRI), CSIR, Lucknow 226001, India
b Microbiology Division, Central Drug Research Institute (CDRI), CSIR, Lucknow 226001, India
c Molecular and Structural Biology Division, Central Drug Research Institute (CDRI), CSIR, Lucknow 226001, India
d Sophisticated Analytical Instruments Facility, Central Drug Research Institute (CDRI), CSIR, Lucknow 226001, India
e Pharmacokinetics and Metabolism Division, Central Drug Research Institute (CDRI), CSIR, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The alarming resurgence of tuberculosis (TB) underlines the urgent need for development of new and
potent anti-TB drugs. Towards this goal we herein report the design and synthesis of 2,3-dideoxy hex-2-
enopyranosid-4-uloses as promising new anti-tubercular agents. These easily accessible, small molecules
were found to exhibit in vitro activity against Mycobacterium tuberculosis H37Rv in a MIC range of
Received 12 August 2010
Received in revised form
15 February 2011
Accepted 1 March 2011
Available online 8 March 2011
0.78
fication of compound 5g (S007e724) which on the basis of low MIC (0.78
1.56 g/mL-MDR, SDR strains of M. tuberculosis; 0.78 g/mL-inhibition of intracellular replication of
M. tuberculosis) and SI value of 13.5 has been identified as a promising lead molecule.
Ó 2011 Elsevier Masson SAS. All rights reserved.
m
g/mL to 25
m
g/mL. A detailed SAR study on these hex-2-enopyranosid-4-uloses led to the identi-
m
g/mL-M. tuberculosis H37Rv;
m
m
Keywords:
Anti-tubercular agents
2,3-Dideoxy hex-2-enopyranosid-4-uloses
Modified deoxy sugars
Small molecules
Non-natural sugars
a, b-Unsaturated keto system
1. Introduction
latent TB, shortened duration of therapy, reduced toxicity and rapid
mycobactericidal mechanism of action [5,7,8]. This paradigm shift
in chemotherapeutic strategies has drawn attention towards the
pathogens’ outer cell wall and membranes as an attractive target
for exploring new drugs [9,10]. Mycobacterial cell wall possesses in
its structure complex polysaccharides lipoarabinomannan and
arabinogalactan [11,12]. The inhibition of the glycosyltransferases
involved in their biosynthesis could lead to the disturbance of the
cell wall biosynthesis [13,14]. Sugar-derived molecules are consid-
ered good candidates for this type of drugs which are thought to act
by interfering with the cell wall biosynthesis of Mycobacterium
tuberculosis [13e19].
Deoxysugars, a distinct class of carbohydrates has received
special attention during the past decades due to their unique pro-
perties [20]. Moreover, current efforts are directed mainly towards
the development of small molecules as drugs which are in many
ways more attractive than large molecules such as antibodies and
proteins. They are more stable, more active, can be easily formu-
lated, have a broader array of dosage forms, have greater potential
for bio-availability and their synthesis can be easily developed
Tuberculosis (TB), a chronic bacterial infection is a leading cause
of mortality worldwide from a single infectious agent [1e3]. The
emergence of multi-drug resistant TB (MDR-TB) and of late exten-
sively drug resistant TB (XDR-TB) along with the problem of co-
infection with HIV has made the threat from TB very serious raising
the spectre that TB may become incurable once again [4e6]. The
presently used anti-TB drugs were introduced more than 40 years
ago [7] when the problem of XDR-TB, MDR-TB and co-infection
with HIV did not exist. Thus, to effectively combat the menace of TB
in its present manifestation there is an urgent need for develop-
ment of new anti-TB agents that have unique mechanisms of action
from presently used drugs with improved properties such as enh-
anced activity against MDR and XDR strains, effectiveness against
* Corresponding author. Tel.: þ91 522 2612411; fax: þ91 522 2623405.
** Corresponding author. Tel.: þ91 522 2620908.
E-mail addresses: drranjana@yahoo.com (R. Srivastava), akshaw55@yahoo.com
(A.K. Shaw).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.002

2218
M. Saquib et al. / European Journal of Medicinal Chemistry 46 (2011) 2217e2223
[21,22]. Since TB is primarily a disease of the poor [1] so cost of
treatment is a major issue in the eradication of TB [23]. Thus smaller
the drug molecule, easier would be its synthesis and the lesser its
cost. A major stumbling block in the development of carbohydrate
based drugs has been their rapid breakdown in the bloodstream by
glycosidases before they can show their therapeutic effect. This
pharmacokinetic problem can be overcome by using modified non-
natural sugars which may not be recognized by the body’s normal
complement of glycosidases [24,25]. In light of the above discussion
we have been focusing our efforts towards the synthesis of modi-
fied deoxy sugar based anti-tubercular agents [18,19,26e28]. In
continuation of our efforts we herein report the synthesis of a new
series of 2,3-dideoxy hex-2-enopyranosid-4-uloses and their
derivatives, which are easily accessible small molecules exhibiting
better activity profile and lesser toxicity than our previously
reported anti-tubercular agents [19].
OR1
6
3
OR1
O
O
5
O
1
O
a
4
OR
2
HO
OR
R
H
1"
R2
R1
R2
m
5 = Piv
= 4-CNC6H4
= 4-NO2C6H4
a = Me
b = Pr
6
i
= Ac
n
7 = Ts
i
o = 2,4-di-NO2C6H3
d
=
Bu
i
g
= Amyl
i = Decyl
Scheme 2. Reagents and conditions: (a) TiCl4, TBAI, R2CHO, DCM,
ꢀ78 ꢁC / ꢀ30 ꢁC.
5 he8 h,
M. tuberculosis could be detected by either a decline or very small
2. Chemistry
increase of the daily GI output as compared to the control. Strep-
tomycin (6
tive control. The MIC of compounds ranged from 0.78
25 g/mL.
m
g/mL) and Rifampin (2
m
g/mL) were used as posi-
The synthesis were initiated with 3,4,6-tri-O-acetyl-
D
-glucal 1,
mg/mL to
easily obtained from -glucose. Compound 1 was subjected to
D
m
Ferrier rearrangement [29] with different alcohols in the presence
of iodine to furnish 2,3-dideoxy glucopyranoside derivative 2. It
was deacetylated in the presence of sodium methoxide to obtain
dihydroxy derivative 3 followed by allylic oxidation with MnO2 to
obtain hydroxyl enuloside 4. Pivaloyl, acetyl and tosyl protection of
C-6 hydroxy group in 4 yielded C-6 protected enulosides 5, 6 and 7
respectively (Scheme 1) in good yields [30,31].
In order to study the structure activity relationship of
compounds 5, 6 and 7, their C-3 alkylaryl branched derivatives
5am, 5dm, 5gm, 5go, 5im, 6gn, 6go, 6in and 7bn were synthesized
by our earlier reported synthetic protocol involving a highly dia-
stereoselective MoritaeBayliseHillman reaction in the presence of
TiCl4/TBAI in DCM at ꢀ78 ꢁC to ꢀ30 ꢁC (Scheme 2) [30,31].
Further the enulosides 5b and 5g (S007-724) were treated with
37% aqueous soln. of HCHO in THF in the presence of DMAP to
obtain their C-3 hydroxymethyl branched enulosides 8 and 9
(Scheme 3) [31].
4. Result and discussion
4.1. Structure activity relationship
The present work was designed on the basis of the reports of
Georgiadis et al. that substituted-2H-pyran-3(6H)-ones, which are
structurally very similar to 2,3-dideoxy hex-2-enopyranosid-4-
uloses I (Fig. 1), exhibit activity against gram positive bacteria
[32,33] as well as our previous report on the anti-tubercular activity
of C-3-alkyl and -alkylaryl 2,3-dideoxy hex-2-enopyranosides [19].
The above reports led us to the idea that simple 2,3-dideoxy hex-2-
enopyranosid-4-uloses I may also exhibit anti-tubercular activity.
To test our hypothesis, 5b, the precursor of our previously
reported most active C-3 branched 2,3-dideoxy hex-2-enopyr-
anosid-4-ulose 5bm (Fig. 2) [19], was evaluated for its in vitro anti-
tubercular activity. We found that the compound 5b exhibited one
fold better anti-tubercular activity at MIC of 3.125
mg/mL compared
3. Biological evaluation
to that of 5bm (MIC-6.25 g/mL) [34e36]. This result prompted us
m
to synthesize a series of 2,3-dideoxy hex-2-enopyranosid-4-uloses
(Scheme 1) and carry out a detailed SAR study in order to delineate
the structural features associated with highest activity and least
toxicity.
First enulosides 4b, 6b and 7b having same substituent at
anomeric position (OiPropyl) but different substituents at C-6, were
evaluated for their anti-tubercular activity. It was found that 4b
having an unprotected hydroxyl group at C-6 showed a moderate
All the compounds were evaluated for their in vitro anti-tuber-
cular activity by BACTEC radiometric method using M. tuberculosis
H37Rv as test strain for direct determination of the minimum
inhibitory concentration (MIC). The assay grows M. tuberculosis in
7H12 medium containing 14C labeled palmitic acid as substrate and
14CO2 is produced. The amount of 14CO2 detected reflects the rate
and amount of growth occurring in the vial and is expressed in term
of the “Growth Index” (GI). On addition of an anti-tubercular drug
to the medium, suppression of growth of the test organism
MIC of 25
mg/mL while 6b and 7b having an acetyl and tosyl group
respectively at C-6 exhibited a good MIC value of 3.125
mg/mL that
was same as that reported for 5b having a pivaloyl group at C-6.A
similar trend was also observed in the case of other enulosides
bearing different alkoxy groups at C-1. These results clearly indi-
cated that the activity of any enuloside was enhanced when an acyl
OH
5
6
OH
OAc
O
OAc
O
4
O
O
a
O
b
1
c
OAc
OAc
OH
OAc
OR
2
3
OR
OR
2
3
4
1
d
R1
R
OPiv
O
OPiv
5 O
OPiv
O
6
i
i
t
a
b
c
d
e
= Methyl, = Propyl, = Butyl, = Butyl, = Butyl,
f = Pentyl, g = Amyl, h = Hexyl, i = Decyl;
j = Cyclopropylmethyl; k = 4-Methoxyphenyl
5 = Piv
O
O
O
a
b
1
i
6 =
Ac
OR1
5
4
6
7 = Ts
O
O
i
OR
1
O Amyl
2
OR
3
4
HO
AcO
1"
i
OR
8 R = Pr
10
5b
5g
3
2
i
9
R = Amyl
Scheme 1. Reagents and conditions: (a) ROH, THF, I2, 3 h-5 h, N2 atm., RT; (b) NaOMe,
MeOH, 1 h-2 h, RT; (c) MnO2, CHCl3, 20 h-40 h, RT; (d) (CH3)3CCOCl/(CH3CO)2O/TsCl,
Pyridine/DCM, 6 h-48 h, ꢀ30 ꢁCe5 ꢁC.
Scheme 3. Reagents and conditions: (a) 37% aq. HCHO, DMAP, THF, ꢀ5 ꢁC, 12 he16 h;
(b) (CH3CO)2O, Pyridine, 0 ꢁC / 5 ꢁC, 12 h.

M. Saquib et al. / European Journal of Medicinal Chemistry 46 (2011) 2217e2223
2219
Table 1
O
In vitro activity of the synthesized 2,3-dideoxy hex-2-enopyranosides and 2,3-
dideoxy hex-2-enopyranosid-4-uloses against M. tuberculosis H37Rv.
OR1
O
O
O
O
R1
OR
O
R1
OR1
O
OR1
O
O
OR
R = alkyl; R1 = aryl
OR
I
HO
OR
R = alkyl, aryl; R1 = alkyl, acyl, tosyl
OR
4a - 7b
3g, 3i
Substituted-2H-pyran-3(6H)-ones
2,3-Dideoxy hex-2-enopyranosid-4-uloses
Fig. 1. Substituted-2H-pyran-3(6H)-ones which are structurally very similar to 2,3-
dideoxy hex-2-enopyranosid-4-uloses I, exhibit activity against gram positive bacteria.
Entry Compd. Code
R
R1
MIC (mg/mL)
Inactivea
Inactive
25
25
12.5
25
6.25
1
2
3g
3i
iso-Amyl
Decyl
Methyl
H
H
H
H
H
H
H
H
H
H
or tosyl group was introduced at C-6. Next the role of the group
attached to C-1 (anomeric carbon) of the enulosides was scruti-
nized. It is well documented that biological activity of alcohols,
including anti-bacterial activity showed regularities of increase and
decrease depending on the structure of the alkyl substituent
[37e41]. On the whole lengthening of the carbon chain causes an
increase in pharmacological effects, but further lengthening brings
about a parabolic decrease in activity [37e42]. Branching raises
activity and so does transition from primary to tertiary alkyl alco-
hols through secondary in an isomeric series [41]. Similar type of
effect has also been seen in case of anti-tubercular activity as was
evident from the SAR studies leading to the discovery of etham-
butol [43]. In the light of these reports we decided to explore the
effect of the alkoxy substituent at C-1 on anti-tubercular activity of
the enuloside. A series of enulosides having different alkoxy
substituents (OMethyl, OiPropyl, OButyl, OiButyl, OtButyl, OPentyl,
OiAmyl, OHexyl, ODecyl) at C-1 were synthesized (Scheme 1) and
evaluated for their anti-tubercular activity (Table 1) for a compar-
ative study of their anti-tubercular activities with their precursor
C-3 unsubstituted enulosides.
It was found that enulosides having the same substituent at C-6
showed a general increase in activity on moving from one carbon
chain (OMethyl) to five carbon chain (OPentyl and OiAmyl) at C-1.
Further lengthening of the carbon chain led to a decrease in activity
and it was lowest for the enuloside bearing a ten carbon ODecyl
substituent at C-1 (Fig. 3, Table 1). The enulosides (5g and 6g) having
an OiAmyl substituent at C-1 were found to be the most active.
To further explore the effect of the substituent at C-1 on anti-
tubercularactivity,compounds4j and5jhavingacyclopropylmethoxy
[44,45] and compounds 4k and 5k having a 4-methoxyphenyl group
[46,47] respectively at C-1 were synthesized (Scheme 1). However,
evaluation of their anti-tubercular activity revealed that the MIC’s
of the most active cyclopropylmethoxy containing enuloside 5j and
3
4
5
6
7
8
4a
4b
4d
4e
4g
4i
iso-Propyl
iso-Butyl
tert-Butyl
iso-Amyl
Decyl
Cyclopropylmethyl
4-Methoxyphenyl
Methyl
12.5
25
9
4j
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4k
5a
5b
5c
5d
5e
5f
5g
5h
5i
Inactive at 25 mg/mL
Pivaloyl 6.25
Pivaloyl 3.125
Pivaloyl 3.125
Pivaloyl 3.125
Pivaloyl 6.25
Pivaloyl 1.56
Pivaloyl 0.78
Pivaloyl 3.125
Pivaloyl 12.5
iso-Propyl
n-Butyl
iso-Butyl
tert-Butyl
n-Pentyl
iso-Amyl
n-Hexyl
Decyl
5j
Cyclopropylmethyl Pivaloyl 3.125
4-Methoxyphenyl
Methyl
iso-Propyl
tert-Butyl
iso-Amyl
5k
6a
6b
6e
6g
6i
Pivaloyl 6.25
Acetyl
Acetyl
Acetyl
Acetyl
Acetyl
Tosyl
6.25
3.125
6.25
0.78
6.25
3.125
2
Decyl
iso-Propyl
7b
Rifampin
Streptomycin
6
a
Inactive at a MIC of 50 mg/mL unless otherwise indicated.
to three folds lower than that of their corresponding precursor
enulosides (Table 2). Interestingly, a similar correlation between
anti-tubercular activity and chain length of the OAlkyl substituent
at C-1, as noticed in the case of the precursor hex-2-enopyranosid-
4-uloses was also observed here (Table 2).
30.0
28.5
27.0
25.5
24.0
22.5
21.0
19.5
18.0
16.5
15.0
13.5
12.0
10.5
9.0
R = H
R1 = Piv
4-methoxyphenyl containing enuloside 5k were 3.125
mg/mL and
Me (4a)
i
R11 = Ac
Propyl
(4b)
6.25
m
g/mL respectively, which were much higher than the MIC of the
alicyclic OiAmyl substituent containing enuloside 5g (Table 1).
We next synthesized a series of C-3 alkylaryl 2,3-dideoxy hex-2-
enopyranosid-4-ulose derivatives (Scheme 2) for a comparative
study of their anti-tubercular activities with their precursor enu-
losides (Table 2).
OR1
O
O
It was found that the anti-tubercular activities of the C-3 alky-
laryl 2,3-dideoxy hex-2-enopyranosid-4-uloses were generally one
Decyl (4i, 5i)
Decyl (6i)
i
Alkyl
O
Butyl
(4d)
i
Amyl (4g)
Me (5a, 6a)
7.5
OPiv
6.0
O
O
i
4.5
Butyl (5d)
i
3.0
Propyl
i
O Pr
1.5
HO
(5b, 6b)
i
Amyl (5g, 6g)
0.0
H
0
1
2
3
4
5
6
7
8
9
10 11
Carbon atoms in alkoxy chain at C-1 pos. of enuloside
CN
Fig. 3. Correlation of anti-tubercular activity of 2,3-dideoxy hex-2-enopyranosid-4-
Fig. 2. Structure of compound 5bm.
uloses with length of alkoxy chain at C-1 position.

2220
M. Saquib et al. / European Journal of Medicinal Chemistry 46 (2011) 2217e2223
Table 2
OH
OTBDMS
CN
In vitro activity of the synthesized C-3 branched 2,3-dideoxy hex-2-enopyranosid-4-
uloses against M. tuberculosis H37Rv.
O
O
O
O
O
b,c,d
HO
i
i
OR1
O
O
11
i
O Amyl
O Amyl
O Amyl
12
4g
e
OR
R3O
H
CN
O
R2
O
i
O Amyl
13
Entry Compd.
Code
R
R1
R2
R3
MIC
g/mL)
Scheme 4. Synthesis of enulosides having different substituent at C-6. Reagents and
conditions: (a) TBDMSCl, Et3N, DCM, 0 ꢁC / 5 ꢁC, 40 h; (b) TsCl, Pyridine, DCM,
ꢀ30 ꢁC / 5 ꢁC, 24 h; (c) NaBH4, CeCl3$7H2O, EtOH, 0 ꢁC / 5 ꢁC, 50 min; (d) NaCN,
DMF, 100 ꢁC-110 ꢁC, 4 h; (e) MnO2, CHCl3, 16 h, RT.
(
m
1
2
3
4
5
6
7
8
5am
5 dm
5gm
5go
5im
6gn
6go
6in
7bn
8
Methyl
iso-Butyl
iso-Amyl
iso-Amyl
Decyl
iso-Amyl
iso-Amyl
Decyl
iso-Propyl Tosyl
iso-Propyl Pivaloyl
iso-Amyl
iso-Amyl
Pivaloyl 4-cyanophenyl
Pivaloyl 4-cyanophenyl
Pivaloyl 4-cyanophenyl
Pivaloyl 2,4-dinitrophenyl
Pivaloyl 4-cyanophenyl
Acetyl
Acetyl
Acetyl
H
H
H
H
H
H
H
H
H
H
H
12.5
6.25
6.25
6.25
12.5
3.125
6.25
25
hydrogenated derivative of 5b was also found to be inactive
(Scheme 5). From the above observations it can be argued that the
presence of the C-4 keto group and the 2,3-double bond in the
pyran ring (a, b-unsaturated keto system) were essential for anti-
tubercular activity of these compounds [48e52].
The SAR studysuggested that the anti-tubercular activity of these
hex-2-enopyranosid-4-uloses may be resulting from the presence of
Michael acceptors in the form of a, b-unsaturated keto system in
4-nitrophenyl
2,4-dinitrophenyl
4-nitrophenyl
4-nitrophenyl
H
H
H
9
6.25
25
25
10
11
12
9
10
Pivaloyl
Pivaloyl
Acetyl Inactive
Rifampin
Streptomycin
2
6
their structure, as is well documented in literature that the biolog-
ical activities [48e51], including anti-bacterial activity [48] of
a number of a, b-unsaturated carbonyl structural element contain-
To further elucidate the role of branching at C-3 position of these
enulosides, C-3 hydroxymethyl branched derivatives 8 and 9 of the
highly active hex-2-enopyranosid-4-uloses 5b and 5g respectively
(Scheme 3) were synthesized [31]. A sharp decrease in anti-
tubercular activity was observed in case of both the C-3 hydrox-
ymethyl branched derivatives 8 and 9 (Table 2).
ing compounds seem to originate from their reaction with nucleo-
philes bya Michael-type addition [48e51]. In addition the SAR study
also seems to suggest a role for the conformation of the enone
moiety in the anti-tubercular activity of these enulosides [52].
The above argument was supported by the observation that
when enuloside 5g was subjected to epoxidation (Scheme 5), the
resulting epoxy derivative 16 was found to exhibit moderate anti-
Acetylation of the hydroxymethyl group in 9 resulted in C-3
acetoxymethyl derivative 10 which was found to be inactive at the
activity at MIC of 12.5 mg/mL unlike in the case of hydrogenation of
concentration of 50
mg/mL (Scheme 3, Table 2). This result was
5g wherein the resulting saturated derivative 15 became inactive.
This result could be rationalized in terms of similar conformation of
the enone moiety in 5g and 16 [52].
contrary to the results obtained in the case of acetylation of the C-5
hydroxymethyl group in enulosides 4 (Scheme 1, Table 1, compounds
of series 6).
The above results clearly showed that introduction of any sub-
stituent at C-3 position of the enulosides 5a, 5d, 5g, 5i, 6g, 6i and 7b
invariably led to a decrease in the anti-tubercular activity though
the quantum of decrease may be related to the nature of the C-3
substituent.
Once the correlation between anti-tubercular activity and the
substituents at C-1 and C-3 of the enulosides was established, we
carried further modifications at C-6 position of the enulosides to
understand more precisely the SAR associated with the substituent
at C-6. When an OTBDMS (ether) group was introduced at C-6
position of 4g (the most active 6-OH containing enuloside) to
obtain compound 11 (Scheme 4), the anti-tubercular activity
4.2. In vitro toxicity evaluation
The most active compounds 5b, 5c, 5d, 5f, 5g, 5h, 5j, 6b, 6g, 7b
and 6gn were tested for their cytotoxicity (Table 4) in an in vitro
model for toxicity with Vero monkey kidney cells originally
obtained from ATCC (ATCC CCL-81) using Resazurin assay as
described in the experimental section below [53,54]. The data from
this toxicity testing and MIC values were used to calculate a selec-
tivity index (SI), the ratio of CC50: MIC (Table 4), an important
Table 3
Variation of anti-tubercular activity of enulosides with the nature of C-6 substituent.
dropped to 12.5
into 2,3,6-trideoxy hex-2-enopyranosid-4-ulose 13 (Scheme 4),
activity got reduced to 12.5 g/mL (Table 3).
mg/mL (Table 3). Likewise when 4g was converted
R1
m
O
O
From the above study it became clear that the C-6 substituent
had a prominent role in modulating the anti-tubercular activity in
the title compounds with the enulosides having an acyl group at
C-6 exhibiting the best anti-tubercular activity (Table 1, Table 3).
Finally we probed the role of C-4 keto group and the double
bond between C-2 and C-3 towards the anti-tubercular activity of
these enulosides (Scheme 5). For this study we chose the most
active hex-2-enopyranosid-4-ulose 5g. Reduction of the C-4 keto
group yielded its 4-hydroxy derivative 14 which was found to be
inactive (Scheme 5). When the double bond between C-2 and C-3 in
5g was hydrogenated, the resulting saturated compound 15 was
found to be inactive (Scheme 5). Likewise compound 17, the
OR
Entry
Compd. Code
R
R1
MIC (
6.25
12.5
12.5
0.78
0.78
2
6
mg/mL)
1
2
3
4g
11
13
5g
6g
iso-Amyl
iso-Amyl
iso-Amyl
iso-Amyl
iso-Amyl
OH
OTBDMS
CN
OPivaloyl
OAcetyl
Entry 17, Table 1
Entry 25, Table 1
Rifampin
Streptomycin

M. Saquib et al. / European Journal of Medicinal Chemistry 46 (2011) 2217e2223
2221
250
OPiv
O
OPiv
O
OPiv
O
O
O
O
a
b
HO
Control
1x MIC
2x MIC
i
O Amyl
i
14
O Amyl
i
15
200
150
100
50
5g
O Amyl
c
OPiv
O
O
i
O Amyl
16
OPiv
OPiv
O
O
O
O
d
i
O Pr
17
5b
i
O Pr
Scheme 5. Reagents and conditions: (a) NaBH4, CeCl3$7H2O, EtOH, 0 ꢁC / 5 ꢁC,
70 min; (b) H2, Pd/C, RT, normal pressure, 80 min; (c) 30% aq. H2O2, 1M NaOH soln.,
MeOH, RT, 30 min, ꢀ10 ꢁC / 0 ꢁC; (d) H2, Pd/C, RT, normal pressure, 90 min.
0
0
6
Days
Table 4
Fig. 4. Graph showing intracellular activity of 5g against M. tuberculosis in terms of
Calculation of Selectivity Index (SI) for the most active compounds (MIC ꢃ 3.125
Growth Index (GI).
m
g/mL).
described in experimental section below. Lysates from 0 and 6 days
of control and test samples were inoculated in BACTEC vials and
growth index (GI) was monitored over 8 day period [55]. As shown
in Fig. 4, the GI rose to 225 in control without the compound, while
at 1ꢂ and 2ꢂ MIC concentration, GI did not rise beyond 10 over the
same period (Fig. 4). The result clearly demonstrated effective
inhibition of intracellular replication of M. tuberculosis within
mouse macrophages at 1ꢂ and 2ꢂ MIC concentrations.
Compound
MIC (
m
g/mL)
MW
MIC (
m
M)
CC50
(
m
M)
SI
1.09
5b
5c
5d
5f
5g
5h
5j
6b
6g
7b
6gn
3.125
3.125
3.125
1.56
270
284
284
298
298
312
282
228
256
340
407
11.5
11
11
5.23
2.65
10.01
11
13.7
3
12.5
42.46
28.16
7.38
36
4.80
77.16
104.4
19.8
13.4
14.3
3.85
2.56
1.41
13.50
0.48
6.96
7.60
6.60
1.40
1.80
0.78
3.125
3.125
3.125
0.78
3.125
3.125
9.19
7.6
5. Conclusion
In summary, we have reported the design and synthesis of 2,3-
dideoxy hex-2-enopyranosid-4-uloses as potentially useful, new
anti-tubercular agents. These small molecules have shown prom-
ising in vitro anti-tubercular activity against virulent H37Rv strain
selection criteria for moving the compounds forward into advanced
in vitro and in vivo studies.
4.3. Detailed biological study on compound 5g (S007-724)
of M. tuberculosis in a MIC range of 25
mg/mL to 0.78 mg/mL.
A detailed SAR study was carried out to understand the structural
basis of anti-tubercular activity of the title compounds and guide
the future development of even more potent 2,3-dideoxy hex-2-
enopyranosid-4-uloses based anti-tubercular entities. It was rev-
ealed that the substituents at C-1 and C-6 were playing a pivotal
role in modulating the activity of these molecules. It was also found
The compound 5g was identified for further study on the basis
of MIC of 0.78
mg/mL and selectivity index (SI) value of 13.50
(Table 1, Table 4).
4.3.1. Evaluation of in vitro activity against MDR, SDR and clinical
strains of M. tuberculosis
The compound 5g was found to exhibit in vitro activity against
MDR (Multi Drug Resistant), SDR (Single Drug Resistant) and clin-
that the presence of
for anti-tubercular activity of these compounds. One of the enulo-
side 5g, on the basis of low MIC (0.78 g/mL-M. tuberculosis H37Rv;
1.56 g/mL-MDR, SDR strains of M. tuberculosis; 0.78 g/mL-inhi-
a, b-unsaturated carbonyl system was essential
m
ical strains of M. tuberculosis at a MIC of 0.78e1.56
mg/mL as
m
m
determined by BACTEC radiometric detection method (Table 5).
bition of intracellular replication of M. tuberculosis) and SI value of
13.5 has been identified as a promising lead molecule.
These results clearly points to the potential usefulness of the
title 2,3-dideoxy hex-2-enopyranosid-4-uloses as lead structures
for the development of cost-effective and potent new anti-tuber-
cular drugs.
4.3.2. Determination of in vitro efficacy of 5g in macrophages
The compound 5g was tested for in vitro efficacy in murine
macrophage cell line J774A.1 at 1ꢂ and 2ꢂ MIC concentrations as
Table 5
In vitro activity of 5g against SDR, MDR and clinical strains of M. tuberculosis.
6. Materials and methods-biology
M. tuberculosis strain
Susceptibility or resistance
to anti-tubercular drugs
MIC (mg/mL)
6.1. Bacterial strains
433 (SDR)
INHr
1.56
1.56
1.56
1.56
0.78
m
g/mL
g/mL
g/mL
g/mL
g/mL
TB-2506/06 (SDR)
2643 (MDR)
TB-1678/05 (MDR)
2280
EMBr
m
m
m
m
Rifr INHr
Rifr INHr
Drug sensitive
M. tuberculosis H37Rv and clinical strains of M. tuberculosis iso-
lated from TB patients were employed to determine anti-tubercular
activity of compounds. Clinical strains obtained from Dr. Sarman

2222
M. Saquib et al. / European Journal of Medicinal Chemistry 46 (2011) 2217e2223
Singh, All India Institute of Medical Sciences, New Delhi were
characterized biochemically and by 16S rRNA sequencing [56].
M. tuberculosis without drug. The medium was replaced every day
at same time with complete RPMI containing 1ꢂ and 2ꢂ MIC test
compound. Macrophage cells were lysed by the addition of 100 mL
6.2. BACTEC radiometric susceptibility assay
of 0.05% SDS. 100 mL lysate was sampled from three wells on day
0 and day 6 for control, test compounds at 1ꢂ MIC and 2 ꢂ MIC. The
lysate was inoculated in BACTEC vials and growth was monitored.
The protocol for macrophage infection has been described earlier
[54,55].
H37Rv strain of M. tuberculosis was grown in BACTEC 12 B vial
until the GI reached around 300. Test compounds were dissolved in
DMSO. Lyophilized Rifampin, Streptomycin and Isoniazid (Becton
Dickinson) were reconstituted as per instructions in manual in
deionized water and 0.1 mL was added into 12B medium. The final
concentration of streptomycin, isoniazid, ethambutol and rifampin
Acknowledgments
were 6, 0.4, 7.5 and 2 mg/mL respectively. Test compounds were
The authors are thankful to Sophisticated Analytical Instrument
Facility (SAIF), CDRI Lucknow, India for providing spectral data and
Mr. Anup K. Pandey for technical assistance. MS, IH, SS and PS thank
Council of Scientific and Industrial Research (CSIR), New Delhi,
India for financial support. VKS thanks Department of Biotech-
nology (DBT), New Delhi, India for financial assistance. CDRI
communication no. 7847
added at varied concentrations in 0.1 mL into 12B medium. 0.1 mL
of the bacterial suspension (GI w300) was added into each of the
BACTEC 12B vial containing the drug. The control vial contained
dilute M. tuberculosis suspension 1:100 prepared by transferring
0.1 mL of the suspension into 9.9 mL of special diluting fluid and
inoculating 0.1 mL of this dilution into the control 12B vial. The vials
were incubated at 37 ꢁC ꢄ 1 ꢁC and read every day at the same time
of day on BACTEC instrument until a GI of 30 or more is achieved in
the control vial.
Appendix. Supplementary data
The difference in the GI values from the previous day and
designated as
DGI was determined for all samples and compared
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.03.002.
with GI of the control vial. If the GI was less in the drug vial than
D
the control, the population was susceptible; if more, it was resis-
tant. MIC of a compound was defined as the lowest concentration
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